Steroidogenesis in the zona glomerulosa of the adrenal cortex II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including microsomes, described above, and mitochondria. The amount of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40–50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20–30 sec of incubation with deoxycholate.The content of RNA, phospholipids, and cytochromeb ₅ in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.     

Isolation of bovine adrenal cortex mitochondria from the zona glomerulosa

Isolation procedures for the bovine adrenal cortex mitochondria from the zona glomerulosa are described. Special care was taken to avoid contamination of the mitochondria from the zona fasciculata.     

Structural studies of the adrenal zona glomerulosa and renal juxtaglomerular apparatus in pregnant sheep

The ultrastructural changes in the adrenal zona glomerulosa and renal juxtaglomerular apparatus have been examined during normal pregnancy in sheep. As pregnancy progressed, increasing numbers of cells in the adrenal zona glomerulosa displayed mitochondria with straight tubular "rod-like" structures replacing their normal lamelliform cristae; groups of cells showing these mitochondrial changes were predominantly located in the middle and superficial regions of the zona glomerulosa, but at all stages remained interspersed with cells with apparently normal mitochondria. In the same animals, the renal juxtaglomerular index was raised, reflecting an increase in renin storage, and juxtaglomerular myoepithelioid cells showed increased numbers of cytoplasmic granules, but no apparent increase in granular endoplasmic reticulum and Golgi profiles; there were no distinguishing morphological changes in juxtaglomerular peripolar cells. These findings provide morphologic evidence of stimulation of the adrenal zona glomerulosa in association with increased juxtaglomerular renin storage during pregnancy. The mitochondrial changes observed in an increasing proportion of cells in the zona glomerulosa closely resemble those seen in sodium-depleted animals, and may reflect the altered steroidogenic capacity of the adrenal gland in pregnant sheep. The finding of groups of cells displaying altered mitochondria lying next to cells with normal mitochondria suggests the presence of cells with different sensitivities to stimuli for aldosterone production or may indicate the presence of different cell types in the zona glomerulosa responding to different stimuli.     

Intramitochondrial bodies in sheep adrenal zona glomerulosa cells

Summary Relatively large, mostly rounded, very electron dense intramitochondrial bodies in adrenal zona glomerulosa cells of sheep are described and their nature and connection to protein in the mitochondria discussed. The so called azocarmine granules seen in the light microscope may be identical with the intramitochondrial bodies in the zona glomerulosa cells.     

Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex

An assumption that the aldosterone-synthesizing enzyme exists only in zona glomerulosa cells apparently contradicts our recent findings that a purified bovine adrenocortical cytochrome P-45011 beta catalyzes the aldosterone formation and the enzyme exists in both zones of the adrenal cortex. To gain more insight into the zone specificity of aldosterone production, the aldosterone-synthesizing activity of mitochondria prepared from the isolated zones of adrenal cortex of various animal species was investigated. The intact mitochondria from the bovine or porcine zonae fasciculata-reticularis could not produce aldosterone whereas those from the zona glomerulosa produced it at a significant rate. When the mitochondria from the zonae fasciculata-reticularis were solubilized by the addition of cholate, they produced aldosterone from corticosterone at a rate comparable to that of those from the zona glomerulosa. The presence of specific factor(s) in the zonae fasciculata-reticularis mitochondria inhibiting expression of the aldosterone synthetic activity is discussed. The mitochondria of the rat zonae fasciculata-reticularis could hardly catalyze aldosterone synthesis under the detergent-solubilized conditions, whereas those of the zona glomerulosa could. Immunoblot analysis revealed that the mitochondria of the zonae fasciculata-reticularis contained a protein of Mr 51,000 which was immunocrossreactive with a monoclonal antibody directed against P-45011 beta, whereas those of the zona glomerulosa contained two immunocrossreactive proteins of Mr 51,000 and 49,000. These results suggest that in the case of rat adrenal cortex, a specific aldosterone-synthesizing enzyme exists in the zona glomerulosa.     

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria.     

An ultrastructural stereologic study of aldosterone secreting adrenal adenomas and of adjacent zona glomerulosa

The ultrastructure of four aldosterone secreting adenomas and of the adjacent zona glomerulosa has been described by the use of stereological techniques. Adenomatous cells (about 2800 microns 3 in volume) invariably displayed a striking abundance of lipid droplets, which occupied about 30% of the cytoplasm. Mitochondria prevalently contained tubulo-lamellar or lamellar cristae, but some cells exhibited organelles with vesicular cristae. Smooth endoplasmic reticulum (SER) was not very abundant. Small lipofuscin-pigment granules were frequently seen and in a few cells they were exceedingly numerous. Zona glomerulosa cells were smaller (about 950 microns 3 in volume) and possessed mitochondria with typical tubulo-lamellar cristae, a plentiful SER and few lipid droplets. They showed the ultrastructural features of elements actively engaged in steroid synthesis. The possible origin of aldosteronoma cells from the zona glomerulosa is discussed.     

Immunocytochemical localization of adrenodoxin in bovine adrenal cortex by protein A-gold technique

To investigate the intracellular localization of the enzymes that are involved in steroid hormone synthesis, an immunocytochemical study of the distribution of adrenodoxin in cells of the bovine adrenal cortex was carried out by the post-embedding immunostaining method and by immuno-cryoultramicrotomy in combination with the protein A-gold technique. Gold particles were seen on the matrix and the inner membrane of all the mitochondria examined, which have typical vesicular or tubulo-vesicular cristae, in parenchymal cells of the zona fasciculata and the zona reticularis. Gold particles were distributed homogeneously among the mitochondria. The density of gold particles on mitochondria in the parenchymal cells of the zona glomerulosa was lower than that of the zona fasciculata, which was similar to that of the zona reticularis. Gold particles were also seen on round, electron-dense intramitochondrial bodies in the parenchymal cells. The intramitochondrial bodies were abundant in the zona glomerulosa and the outer region of the zona fasciculata.     

Steroidogenic characteristics of the adrenal cortex of the mare studied by electron microscopy

The three concentric zones of the horse adrenal cortex (zonae glomerulosa, fasciculata and reticularis) showed marked interpenetration and exhibited a different relative development according to their position in the gland. Whereas the three cortical zones each had a specific histological structure, the ultrastructure of their cells showed a certain qualitative homogeneity. The differences corresponded essentially to the relative abundance of the constituents which are generally considered typical of steroidogeneous cells: mitochondria with vesicular cristae, smooth endoplasmic reticulum and lipid droplets. Their importance increased progressively from the zona glomerulosa to the zona reticularis. In this zone, the presence of gap and septate-like cell junctions, and mitochondria with vesicular cristae in close proximity to a smooth endoplasmic reticulum with numerous dilated tubules, suggested that steroidogenesis may be the most active. Ultrastructural findings were indicative of only quantitative differences between the steroidogenic capacities of the three zones of the mare adrenal cortex.     

Immunocytochemical localization of adrenodoxin in bovine adrenal cortex by protein A-gold technique

Summary To investigate the intracellular localization of the enzymes that are involved in steroid hormone synthesis, an immunocytochemical study of the distribution of adrenodoxin in cells of the bovine adrenal cortex was carried out by the post-embedding immunostaining method and by immuno-cryoultramicrotomy in combination with the protein A-gold technique. Gold particles were seen on the matrix and the inner membrane of all the mitochondria examined, which have typical vesicular or tubulo-vesicular cristae, in parenchymal cells of the zona fasciculata and the zona reticularis. Gold particles were distributed homogeneously among the mitochondria. The density of gold particles on mitochondria in the parenchymal cells of the zona glomerulosa was lower than that of the zona fasciculata, which was similar to that of the zona reticularis. Gold particles were also seen on round, electron-dense intramitochondrial bodies in the parenchymal cells. The intramitochondrial bodies were abundant in the zona glomerulosa and the outer region of the zona fasciculata.Supported by a Grant-in-Aid for Scientific Research on Priority Areas (no. 63635505) from the Ministry of Education, Science and Culture of Japan     

alpha-MSH analogues and adrenal zona glomerulosa function

Recent studies on the control of adrenal zona glomerulosa function and aldosterone secretion have focussed attention on the role of MSH-like peptides. In particular, at low concentrations, alpha-MSH has a specific stimulatory effect on rat adrenal glomerulosa cells. The synthesis of alpha-MSH analogues which have potent and prolonged effects on melanocyte systems offers new methods of examining the specificity of this response. Two peptides were tested in which potential for a beta-turn configuration was stabilised. These were: [Nle4, D-Phe7]-alpha-MSH and the cyclic [Cys4, Cys10]-alpha-MSH. In contrast to their effects on melanocyte systems, only [Cys4, Cys10]-alpha-MSH stimulated glomerulosa cells, and it was equipotent with alpha-MSH, while [Nle4, D-Phe7]-alpha-MSH and shorter fragments had no effect when added alone. [Nle4, D-Phe7]-alpha-MSH, however, augmented the response of cells already maximally stimulated with alpha-MSH and in this respect its actions resembled those of gamma-MSH and related peptides. The augmentation produced by [Nle4, D-Phe7]-alpha-MSH and gamma 3-MSH was not additive when the two peptides were added together with alpha-MSH. The results suggest that the specificity of the alpha-MSH receptors in rat adrenal glomerulosa cells and the peptide structure-function relationships in this system are different from those described for melanocytes.     

Rat atrial natriuretic polypeptide stimulation of adrenal zona glomerulosa cell growth

Rat atrial natriuretic polypeptide (rANP) was found to stimulate [3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in a primary culture in a dose-dependent manner. The minimum effective dose was a very low concentration (10(-12) M of ANP), suggesting that ANP had a physiological effect. These findings are the first indication that ANP possesses growth-stimulating activity with regard to adrenal zona glomerulosa cells.     

Effects of IGF-I on proliferation and steroidogenesis of cultured adrenal zona glomerulosa cells

In a primary culture of bovine adrenal zona glomerulosa cells, insulin-like growth factor I (IGF-I)/somatomedin C stimulated DNA synthesis, as measured by [3H] thymidine uptake, at concentrations of 10(-9) and 10(-7) M. IGF-I also prevented ACTH-induced suppression of [3H] thymidine uptake. IGF-I in no way affected aldosterone secretion during short-term exposure to cultured cells, however. Our findings suggest that IGF-I plays an important role in the proliferation of adrenal zona glomerulosa cells.     

Adrenal zona glomerulosa after hypophysectomy in the Mongolian gerbil

The ultrastructure of the adrenal zona glomerulosa was examined in hypophysectomized female Mongolian gerbils (Meriones unguiculatus). After 3 weeks, the width of the zona glomerulosa increased significantly. As early as 2 weeks, the smooth endoplasmic reticulum became hypertrophic and the Golgi apparatus was well developed. Two populations of coated vesicles were observed with increased frequency in zona glomerulosa cells of hypophysectomized gerbils     

Chronic hypoxia induced ultrastructural changes in the rat adrenal zona glomerulosa

The adrenal cortex plays an important role in adaptation to various forms of stress, including hypoxia. While physiological changes in the aldosterone metabolism during hypoxia have been extensively described, few studies have focused on the morphological changes in the adrenal glands under chronic hypoxia. We studied the ultrastructure of the zona glomerulosa of 6-month-old Wistar rats exposed to chronic normobaric hypoxia. Animals were divided into two groups: control (n=12) and hypoxic (n=12). In this latter group, the animals were kept at 7% O2 concentration after a gradual adaptation (21, 15, 12, 10, 8, 7 vol% O2). The duration of the study was 112 days. In comparison with normoxic rats, body weight and adrenal gland weight of hypoxic animals was significantly reduced by 18.5% (p=0.006) and 14.7% (p=0.001) respectively. The thickness of the zona glomerulosa decreased due to atrophy of cells. The main ultrastructural changes observed were: 1) a decrease in, or complete elimination of, lipid droplet content; 2) a marked increase in lysosome number; and 3) the presence of giant mitochondria. Our findings show that rats fail to adapt to severe chronic hypoxia. The ultrastructural changes in the zona glomerulosa found in the present study could reflect changes in the aldosterone pathway.     

Effect of a water-rich diet on adrenal zona glomerulosa in Gerbillus tarabuli

The objective of this work is to evaluate the aldosterone level and to study the gerbil adrenal zona glomerulosa morphology in the presence of an important water diet content during seven days. Thirty adult gerbils, Gerbilus tarabuli, were randomly assigned to two watering regimes, with fifteen gerbils per group. The first group was the control and the second group was exposed to a water-rich diet during seven days. Thin sections of the adrenal glands were observed in photonic and electron microscopy. Their blood was sampled and analyzed for aldosterone, electrolyte concentrations and hematocrit. Urine was collected after 24hours. A water-rich diet decreased the body weight but increased the hematocrit, total protein, aldosterone concentration and urine volume. In electronic microscopy, the adrenal zona glomerulosa cells present a decrease in lipid compartment and Golgi apparatus zone development. The blood electrolyte concentrations were not changed significantly. These findings indicate that the water-rich diet exerts a potent stimulatory effect on aldosterone secretion by the gerbil adrenal zona glomerulosa through plasma volume perturbation.     

Cytochrome P-45011 beta and P-450scc in adrenal cortex: zonal distribution and intramitochondrial localization by the horseradish peroxidase-labeled antibody method

In an attempt to elucidate the regulation mechanism(s) of adrenocortical steroidogenesis, cytochrome P-450scc and cytochrome P-45011 beta were localized in bovine adrenal glands by the direct peroxidase-labeled antibody method. At the light microscopic level, parenchymal cells of the zona fasciculata and the zona reticularis stained heavily for both cytochromes, while the parenchymal cells of zona glomerulosa stained lightly for both. At the electron microscopic level, these two cytochromes were associated with the matrix side of the inner mitochondrial membranes of parenchymal cells from all three zones of the adrenal cortex. The association of cytochrome P-450 with the inner mitochondrial membrane, in a manner similar to that previously reported for adrenodoxin and adrenodoxin reductase (F Mitani, Y Ishimura, S Izumi, K Watanabe, Acta Endocrinol 90:317, 1979), establishes that the steroid monooxygenase systems exist at this site. The degree of immunocytochemical staining within a single cell varied from one mitochondrion to another: some stained intensely along the entire inner membrane, including the cristae, some stained only along segments of the inner membrane, and some did not stain at all. This heterogeneity in staining was observed in mitochondria stained in situ as well as in isolated mitochondria. These findings suggest that there is a heterogeneity in steroidogenesis among mitochondria contained within a single cell of the adrenal cortex.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Mitochondrial steroid metabolism in the inner and outer zones of the guinea-pig adrenal cortex

Previous investigations have demonstrated that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea-pig adrenal cortex produce far more cortisol than those from the inner zone (zona reticularis). Studies were carried out to compare mitochondrial steroid metabolism in the two zones. Protein and cytochrome P-450 concentrations were similar in outer and inner zone mitochondria. However, the rate of 11 beta-hydroxylation was significantly greater in the outer zone despite the fact that substrates for 11 beta-hydroxylation (11-deoxycortisol, 11-deoxycorticosterone) produced larger type I spectral changes in inner zone mitochondria. The apparent affinities of 11-deoxycortisol and 11-deoxycorticosterone for mitochondrial cytochrome(s) P-450 were similar in the two zones. In both inner and outer zone mitochondria, 11 beta-hydroxylation was inhibited by metyrapone but unaffected by aminoglutethimide. Cholesterol sidechain cleavage activity, measured as the rate of conversion of endogenous cholesterol to pregnenolone, was far greater in outer than inner zone mitochondria. Addition of exogenous cholesterol or 25-hydroxycholesterol to the mitochondrial preparations did not affect pregnenolone production in either zone. Addition of pregnenolone to outer zone mitochondria produced a reverse type I spectral change (delta A 420-390 nm), suggesting displacement of endogenous cholesterol from cytochrome P-450. In inner zone mitochondria, pregnenolone induced a difference spectrum (delta A 425-410 nm) similar to the reduced vs oxidized cytochrome b5 spectrum. A b5-like cytochrome was found to be present in the mitochondrial preparations. Prior reduction of the cytochrome with NADH eliminated the pregnenolone-induced spectral change in inner zone mitochondria but had no effect in outer zone preparations. The results suggest that differences in mitochondrial steroid metabolism between the inner and outer adrenocortical zones account in part for the differences in cortisol production by cells in each zone.